1. Field of the Invention
The present invention generally relates to the regulation of steroid hormone responsive cancer cell growth, and more particularly to compositions and in vitro methods and models for demonstrating secretory immune system immunoglobulin regulation of mucosal epithelial cancer cell growth.
2. Description of Related Art
Steroid Hormone Responsive Tumor Cell Growth
In 1896, a physician named Beatson reported in the medical journal Lancet (Beatson G T (1896) Lancet (Part 1, July 11), 104-107 and Lancet (Part 2, July 18), 162-165) that removal of the ovaries from breast cancer patients slowed or stopped the growth of their tumors. As medical science has moved forward, it is now understood that Dr. Beatson had found that the estrogens made by the ovaries promoted the growth of breast cancers. In the 1940s and 1950s, work by Professor Charles Huggins (Huggins C B and Hodges C V (1941) Cancer Res 1, 293-297; Huggins et al. (1941) Arch Surg 43, 209-223) proved that surgical or chemical castration very substantially reduced the growth of prostate cancers. These results indicated that testicular androgens were important promoters of the growth of tumors of this male accessory organ. In subsequent work, researchers have established that estrogens and androgens act on breast and prostate cancer cells via receptors within the cell nucleus (Tsai M-J and O'Malley B W (1994) Annu Rev Biochem 63, 451-486; Evans R E (1988) Science (Wash D.C.) 240, 889-895). In fact, estrogen receptors are now commonly measured in breast cancer specimens to assist in decisions regarding the most effective therapies for each patient, and chemical and surgical castration are common treatments for prostate cancer. The regulation of estrogen target tissue cell growth has been a topic of dynamic experimental interest for several years (Jensen E V and DeSombre E R (1973) Science (Wash D.C.) 182, 126-134; O'Malley B W and Means A R (1974) Science (Wash D.C.) 183, 610-620). Today, it is generally accepted that estrogen interaction with specific nuclear located DNA binding receptors is necessary to initiate critical cell cycle events (Dickson R B and Stancel G M (2000) J Natl Cancer Inst Monogr No 27, 135-145). It is also highly likely that other non-steroid factors are essential participants in this process (Sirbasku D A (1978) Proc Natl Acad Sci USA 75, 3786-3790; Sirbasku D A (1981) Banbury Report 8, 425-443; Dickson R B and Lippman M E (1987) Endocr Rev 8, 2943; Soto A M and Sonnenschein C (1987) Endocr Rev 8, 44-52). Many of these new regulators fall into the general class of positive acting substances called growth factors (Gospodarowitz D and Moran J S (1976) Annu Rev Biochem 45, 531-558; Goustin A S et al. (1986) Cancer Res 46, 1015-1029). Simply stated, these agents cause cells to undergo cell division and thereby lead to growth. Because the hallmark of cancer is uncontrolled cell division, understanding these molecules and how they act is of vital importance. Other members of this regulatory family include negative acting agents called growth inhibitors (Knabbe et al. (1987) Cell 48, 417-428; de Jong J S et al. (1998) J Pathol 184, 44-52). They block cell division, and because of this, are important targets for new anticancer therapies. A great deal of study has focused on cellular site(s) of estrogen action, and various models have been proposed attempting to explain how estrogen participates with these additional factors to regulate growth.
The relative merits of positive versus negative regulation of cell growth have been debated (Dickson R B and Lippman M E (1987) Endocr Rev 8, 2943; Soto A M and Sonnenschein C (1987) Endocr Rev 8, 44-52). Although the positive direct and positive indirect models (as defined by Soto A M and Sonnenschein C (1987) Endocr Rev 8, 44-52) have received the most attention, the concept of negative regulation has intrinsic appeal because its loss offers a ready explanation for the uncontrolled replication of cancer cells. Factors that negatively regulate cell proliferation are now classified as members of the “tumor suppressor” family (Sager R (1997) Proc Natl Acad Sci USA 94, 952-955). Defining and understanding this family of intracellular and extracellular growth regulators is a primary focus of current cancer research.
A number of years ago, studies were reported which indicated that serum-borne inhibitors, later named “estrocolyones,” had an important if not essential role in steroid responsive cell growth (Soto A M and Sonnenschein C (1987) Endocr Rev 8, 44-52; Soto A M et al. (1992) J Steroid Biochem Mol Biol 43, 703-712; Soto A M et al. (1986) Cancer Res 46, 2271-2275; Soto A M and Sonnenschein C (1984) Biochem Biophys Res Commun 122, 1097-1103; Schatz R W et al. (1985) J Cell Physiol 124, 386-390; Soto A M and Sonnenschein C (1985) J Steroid Biochem 23, 87-94). Estrocolyones appeared to act as estrogen reversible inhibitors of steroid hormone target tissue cell growth. Subsequently, the inhibitor has been variously identified as an unstable Mr 70,000 to 80,000 protein (Soto A M et al. (1992) J Steroid Biochem Mol Biol 43, 703-712), the intact serum albumin molecule (Laursen I et al. (1990) Anticancer Res 10, 343-352; Sonnenschein C et al. (1996) J Steroid Biochem Mol Biol 59, 147-154), two domains of serum albumin (Sonnenschein C et al. (1996) J Steroid Biochem Mol Biol 59, 147-154), and the plasma steroid carrier protein sex hormone binding globulin (SHBG) (Reese C C et al. (1988) Ann N.Y. Acad Sci 538, 112-121; Fissore F et al. (1994) Steroids 59, 661-667; Fortunati N et al. (1993) J Steroid Biochem Mol Biol 45, 435-444). Other investigators also thought it possible that SHBG, as well as the other major plasma steroid hormone carrier protein corticosteroid-binding globulin (CBG), were potential growth regulators independent of their steroid hormone binding capacity. This conclusion was based on the fact that specific cellular membrane receptors have been identified for steroid free CBG and SHBG (Hryb D J et al. (1986) Proc Natl Acad Sci USA 83, 3253-3256; Hryb D J et al. (1990) J Biol Chem 265, 6048-6054) and that binding of SHBG and CBG to cells caused changes to cell growth mediators such as cyclic AMP and protein kinase A (Rosner W (1990) Endocrine Rev 11, 80-91; Fortunati N et al. (1996) Endocrinology 137, 686-692; Rosner W et al. (1991) J Steroid Biochem Mol Biol 40, 813-820; Nakhla A M et al. 153, 1012-1018; Rosner W (1992) J Andrology 13, 101-106).
Nonetheless, the roles of both albumin and SHBG as estrogen reversible serum-borne growth regulators have been challenged by the present Inventor, and others (Soto A M et al. (1992) J Steroid Biochem Mol Biol 43, 703-712; Damassa D A et al. (1991) Endocrinology 129, 75-84). In fact, in one report, SHBG stimulated growth of the androgen responsive ALVA-41 human prostate cancer cell line (Plymate S R et al. (1991) J Steroid Biochem Mol Biol 40, 833-839). In 1997, Sirbasku et al. reported that nearly pure CBG and an approximately 85% homogeneous SHBG-like protein were obtained from horse serum (Sirbasku D A et al. “Serum factor regulation of estrogen responsive mammary tumor cell growth.” Proceedings of the 1997 Meeting of the “Department of Defense Breast Cancer Research Program: An Era of Hope”, (Abstract) pp. 739-740, Washington, D.C., Oct. 31-Nov. 4, 1997) by employing a procedure similar to that described for use with human cord serum (Fernlund P and Laurell C-B (1981) J Steroid Biochem 14, 545-552). The Femlund and Laurell procedure was stated to produce human CBG and SHBG in pure or very nearly pure states using cortisol-agarose affinity chromatography at pH 5.5 followed by Phenyl Sepharose™ chromatography at pH 7.4. Under serum-free defined cell culture conditions, the partially purified SHBG-like fraction obtained by Sirbasku et al. demonstrated progressive inhibition of cell growth in a rat mammary tumor cell line (MTW9/PL2) with increasing concentration of the SHBG-like fraction. Addition of 17 β-estradiol (E2) completely reversed even the maximum inhibition. Sirbasku et al. found that the active SHBG-like fraction contained little or no serum albumin as judged by immunological methods and by standard polyacylamide gel electrophoresis in the presence of reducing agents and sodium dodecyl sulfate (SDS-PAGE) (Laemmli U K (1976) Nature (Lond) 227, 680-685). Although the SHBG-like inhibitor displayed certain immunological similarities to SHBG, it was clearly distinguishable from SHBG based on physiologic, physical and biochemical analyses. Despite its first proposal more than fifteen years ago, the purified estrogen reversible serum-borne inhibitor has yet to be described. Sirbasku et al., as well as others (Soto A M et al. (1992) J Steroid Biochem Mol Biol 43, 703-712), has observed that the estrogen reversible inhibitory activity of serum was very labile during isolation by conventional protein purification methods. Other investigators have used a combination of cortisol affinity chromatorgraphy and an ammonium sulfate precipitation to isolate a cell growth inhibitor from human serum. These studies (Tanji M et al. (2000) Anticancer Res. 20, 2779-2783; Tanji M et al. (2000) Anticancer Res. 20, 2785-2789) describe estrogen inhibition of MCF-7 human breast carcinoma cells that had been maintained at least 3 months in serum-free medium, but no estrogenic effect was observed with normally cultured MCF-7 cells (i.e., cells not long term conditioned to serum-free medium). An isolated steroid-binding protein was stated to mediate an estrogen-dependent inhibition of cell growth. Other serum-borne inhibitors also have been separated from whole serum by diethylaminoethyl (DEAE) chromatography (Dell' Aquila M L and Gaffney E V (1984) J Natl Cancer Inst 73, 397-403). The properties of these inhibitors have not been defined further nor have they been shown to act as estrogen-reversible inhibitors.
Carcinogen-induced rat mammary tumors have been studied extensively as models for the in vivo role of hormones in the induction and growth of breast cancer (Welsch C W (1985) Cancer Res 45, 3415-3443). Despite ample evidence of hormone dependence in vivo, the carcinogen-induced tumors have not yet yielded permanent tissue culture cell lines that show the same responsiveness to steroid hormones in in vitro culture. Typically, cultures initiated from primary tumors very quickly lose hormone responsiveness. Because of this, the earliest endocrine studies were done with organ cultures (Welsch C W and Rivera E M (172) Proc Soc Exp Biol Med 139, 623-626; Lewis D and Hallowes R C (1974) J Endocrinol 62, 225-240; Chan P-C et al. (1976) Proc Soc Exp Biol Med 151, 362-365; Pasteels J-L et al. (1976) Cancer Res 36, 2162-2170) and short-term cultures of dissociated cells (Chan P-C et al. (1976) Proc Soc Exp Biol Med 151, 362-365). Now investigators recognized that those approaches were inadequate. More recently, cell lines have been developed from carcinogen-induced rat mammary tumors (Bennett D C et al. (1978) Cell 15, 283-298; Rudland P S (1987) Cancer Metast Rev 6, 55-83; Webster M K et al. (1990) J Biol Chem 265, 4831-4838; Lichtner R B et al. Cancer Res 51, 5943-5950; Lichtner R B et al. (1995) Oncogene 10, 1823-1832). Although these lines have been useful for investigations related to breast properties, investigators have found that in general they do not display steroid hormone responsiveness in cell culture. To compound the difficulties, most of these lines could not be evaluated for hormone responsiveness in vivo because they were derived from outbred rats. Simply stated, they lack the syngeneic inbred hosts absolutely required for in vivo transplantation.
One of the basic tenets of endocrine physiology is that estrogens and androgens cause coordinate growth of several target tissues (Clark J H et al. (1992) In: Williams Textbook of Endocrinology, 8th Edition, W B Saunders, Philadelphia, pp 35-90). A partial list of estrogen target tissues includes breast, uterus, cervix, vagina, ovary, pituitary, liver, leukocytes and kidney. A partial list of androgen target tissues includes the male reproductive tract (e. g. prostate, epididymus, and testis), kidney, bladder, liver and muscle. Whatever mechanism is proposed to explain sex steroid dependent growth, one would expect it to be applicable to cells from several of the major target tissues.
The history of attempts to demonstrate steroid hormone responsive tumor cell growth in culture has led to two important conclusions. First, demonstration of estrogen and androgen responsive cell growth in culture required the presence of hormone deficient/depleted serum. One of the first studies to demonstrate this requirement was done with human breast cancer cells (Page M J et al. (1983) Cancer Res 43, 1244-1250). Some notable examples of demonstration by others of estrogen responsiveness in serum containing culture include studies with the MCF-7 human breast cancer cells (Lippman M E et al. (1977) Cancer Res 37, 1901-1907; Soto A M and Sonnenschein C (1985) J Steroid Biochem 23, 87-94; Wiese T E et al. (1992) In Vitro Cell Dev Biol 28A, 595-602), the T47D human breast cancer cells (Chalbos D et al. (1982) J Clin Endocrinol Metab 55, 276-283; Schatz R W et al. (1985) J Cell Physiol 124, 386-390; Soto A M et al. (1986) Cancer Res 46, 2271-2275), the ZR-75-1 human breast cancer cells (Darbre P et al. (1983) Cancer Res 43, 349-355), the GH4C1 rat pituitary tumor cells (Amara J F and Dannies P S (1983) Endocrinology 112, 1141-1143), and the H-301 Syrian hamster kidney tumor cell line (Soto A M et al. (1988) Cancer Res 48, 3676-3680). Two reports have proposed that estrogen responsiveness can be observed in serum-free defined medium with ZR-75-1 cells (Allegra J C and Lippman M E (1978) Cancer Res 38, 3823-3829; Darbre P D et al. (1984) Cancer Res 44, 2790-2793). However, in both of those studies, the cells were first incubated for several days in medium supplemented with serum before changing to serum-free defined medium conditions. M Ogasawara and D A Sirbasku previously demonstrated that this approach leaves a problematic serum factor “memory” with cells (Ogasawara M and Sirbasku D A (1988) In Vitro Cell Dev Biol 24, 911-920). When completely serum-free defined medium conditions were applied (Barnes D and Sato G (1980) Nature 281, 388-389; Danielpour D et al. (1988) In Vitro Cell Dev Biol 24, 42-52; Karey K P and Sirbasku D A (1988) Cancer Res 48, 4083-4092; Ogasawara M and Sirbasku D A (1988) In Vitro Cell Dev Biol 24, 911-920; Riss T L and Sirbasku D A (1989) In Vitro Cell Dev Biol 25, 136-142), no growth effects of estrogens were observed. Comparison of the observations in serum-free defined medium versus those in medium with serum led to the second important conclusion. Serum contains a mediator(s) that is required for steroid hormone responsiveness in culture. When the mediator is completely purified and defined chemically, its addition to serum-free defined medium will be expected to provide unequivocal confirmation of its role in hormone dependent cell growth.
The purification of the serum-borne mediator has been a challenging undertaking. Sirbasku et al. originally proposed that estrogens per se were not mitogenic, but instead caused the production of endocrine, paracrine or autocrine “estromedins” that were themselves the promoters of target tissue cell growth (Sirbasku D A (1978) Proc Natl Acad Sci USA 75, 3786-3790; Sirbasku D A (1981) Banbury Report 8, 425-443; Ikeda T et al. (1982) In Vitro 18, 961-979; Sirbasku D A and Leland F E (1982) Biochemical Action of Hormones 9, 115-140; Leland F E et al. In: Cold Spring Harbor Conferences on Cell Proliferation, Volume 9, Books A and B, Growth of Cells in Hormonally Defined Media, Cold Spring Harbor, N.Y., pp 741-750). From 1970 through 1984, estrogenic mitogenic effects were most often not seen in culture. Although some laboratories were reporting positive results in serum containing medium, as cited above, others were at the same time recording negative results using the same or related cell lines (Sirbasku D A (1978) Proc Natl Acad Sci USA 75, 3786-3790; Sirbasku D A and Kirkland W L (1976) Endocrinology 98, 1260-1272; Kirkland W yL et al. (1976) J Natl Cancer Inst 56, 1159-1164; Ikeda T et al. (1982) In Vitro 18, 961-979; Butler W B et al. (1983) Cancer Res 41, 82-88; Edwards D P et al. (1980) Biochem Biophys Res Commun 93, 804-812; Shafie S M (1980) Science (Wash D.C.) 209, 701-702). Part of the problem may have been due to culture conditions (Ruedl C et al. (1990) J Steroid Biochem Mol Biol 37, 195-200; Zugmaier G et al. (1991) J Cell Physiol 141, 353-361) or possibly caused by differences that arose because of variations in the properties of cell lines in different laboratories (Seibert K et al. (1983) Cancer Res 43, 2223-2239). In addition, there are other more technical issues that are well known in this field, have been described in the literature, and which are addressed in more detail elsewhere herein and in subsequent publications (Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev Biol 36, 410-427; Sirbasku D A and Moreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36, 428-446; and Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev Biol 36, 447-464.) Another vital matter has been how “growth” is defined. Sonnenschein and Soto (Sonnenschein C and Soto A M (1980) J Natl Cancer Inst 64, 211-215) have addressed this issue very effectively. To be accepted as valid, sex steroids must cause significant changes in cellular logarithmic growth rates. Elucidation of the nature and activity of the estrogen reversible serum inhibitor(s) continues to be an area of intense experimental interest.
As cited above, A M Soto and C Sonnenschein have proposed that the serum mediator is an estrogen reversible inhibitor they have named estrocolyone. They have alternately described the inhibitor as a pituitary factor (Sonnenschein C and Soto A M (1978) J Steroid Biochem 6, 533-537), α-fetoprotein (Sonnenschein C et al. (1980) J Natl Cancer Inst 64, 1141-1146; Sonnenschein C et al. (1980) J Natl Cancer Inst 64, 1147-1152; Soto A M and Sonnenschein C (1980) Proc Natl Acad Sci USA 77, 2084-2087), a serum protein different than human serum albumin (Soto A M et al. (1992) J Steroid Biochem Mol Biol 43, 703-712), and in a later reversal of this view, stated that estrocolyone 1 (i.e. the serum-borne estrogen reversible inhibitor) was human serum albumin or a combination of two domains of albumin (Sonnenschein C (1996) J Steroid Biochem Mol Biol 59, 147-154). They have also sought the inhibitor as an estrogen-binding glycoprotein different than SHBG using Concanavalin-A chromatography (Reny J-L and Soto A M (1989) J Clin Endocrinol Metab 68, 938-945). The outcome of this effort did not identify the inhibitor. The exact chemical nature of the inhibitor was even further complicated by U.S. Pat. No. 4,859,585 (Sonnenschein) and U.S. Pat. No. 5,135,849 (Soto) describing an inhibitor that was derived from heat inactivated serum depleted of its endogenous estrogens and androgens by a 37.5° C. charcoal-dextran procedure. Alternatively, the inhibitor was obtained from serum by ammonium sulfate precipitation. This inhibitor is said to be useful for in vitro testing of substances of interest for activity as an estrogen or androgen agonist or antagonist using the MCF-7 cell line grown in Dulbecco's modified Eagle minimal essential medium supplemented with 5% (v/v) fetal bovine serum. However, the two above-mentioned U.S. patents do not address the issues of (i) whether there are one or more inhibitors, (ii) what is/are the exact chemical composition of the inhibitor(s), and (iii) what conditions were required to yield the long term stable product(s) necessary for the commercial application of the testing methodology described.
Steroid Hormone Receptors
As the matter stands today, it has not been established beyond doubt which of the many estrogen receptors and/or variants is the one that regulates the estrogen induced mitogenic effect. It is generally assumed that the ERα is the most likely positive growth mediator. Estrogens, androgens, progestins, corticosteroids, mineral steroids, vitamin D, retinoic acid and thyroid hormone receptors all belong to a family of DNA binding intracellular receptors that are activated by binding of the appropriate hormone/ligand (Evans R M (1988) Science (Wash D.C.) 240, 889-895; Giguere V (1990) Genetic Eng (NY) 12, 183-200; Williams G R and Franklyn J A (1994) Baillieres Clin Endocrinol Metab 8, 241-266; Kumar R and Thompson E B (1999) Steroids 64, 310-319; Pemrick S M et al. (1994) Leukemia 8, 1797-806; Carson-Jurica M A et al. (1990), Endocr Rev 11, 201-220; Tsai M J and O'Malley B W (1994) Annu Rev Biochem 63, 451-486; Alberts B et al. (1994) Molecular Biology of The Cell, 3rd edition, Garland Publishing, New York, pp 729-731). The estrogen receptor described in the citations above is now designated the classical estrogen receptor alpha (ERα). Its role in steroid regulated gene expression has been studied extensively and often reviewed (Yamamoto K R (1985) Annu Rev Genet 19, 209-252; Green S and Chambon P (1991) In: Nuclear Hormone Receptors, Academic Press, New York, pp 15-38; Tsai M-J and O'Malley B W (1994) Annu Rev Biochem 63, 451-486; McDonnell D P et al. (1992) Proc Natl Acad Sci USA 89, 10563-10567; Landel C C et al. (1994) Mol Endocrinol 8, 1407-1419; Landers J P and Spelsberg T C (1992) Crit Rev Eukary Gene Exp 2, 19-63; Cavailles V et al. (1994) Proc Natl Acad (Sci USA 91, 10009-10013; Halachmi S et al. (1994) Science (Wash D.C.) 264, 1455-1458; Brasch K and Ochs R L (1995) Int rev Cyto 159, 161-194; Härd T and Gustafsson J-Å (1993) Acc Chem Res 26, 644-650).
It is noteworthy that estrogen resistance in man is caused by a mutation in the ERα (Smith E P et al. N Eng J Med 331, 1056-1061). The most startling fact is that this point mutation (i.e. cytosine→thymidine) generated a premature stop codon, but was not lethal. Although many metabolic abnormalities were noted, development into adulthood was observed without expression of a functional ERα. This fact is further strengthened by the experiments with ERα gene knockout mice (Couse J F and Korach K S (1999) Endocr Rev 20, 358-417). The authors state “the list of unpredictable phenotypes in the α ERKO (estrogen receptor knockout) must begin with the observation that generation of an animal lacking a functional ER α gene was successful and produced animals of both sexes that exhibit a life span comparable to wild-type”. Furthermore, in the review of the ERKO results, it was not possible to conclude that the ERα regulated estrogen responsive cell growth. Indeed, functions normally ascribed to the ERα seemed unaffected. In fact, only relationships to development in tissues such as breast seemed best correlated (Boccchinfuso W P and Korach K S (1997) J Mammary Gland Biol Neoplasia 2, 323-334). The situation with ERKO mice and ERβ is similar (Couse J F and Korach K S (1999) Endocr Rev 20, 358-417). The results from ERβ knockout suggest an indirect role of this receptor via stromal tissue (Gustafsson J-Å and Warner M (2000) J Steroid Biochem Mol Biol 74, 254-248). Certainly a direct growth role for ERβ in breast epithelial cells was not established. The results available from ERKO do not yet provide confidence that either the ERα or the ERβ mediate estrogen responsive cell growth.
There are other pertinent lines of evidence that relate to the role of the ERα and growth. The first is from a study of transfection of estrogen receptor negative cells with the full length functional ERα (Zajchowski D A et al. (1993) Cancer Res 53, 5004-5011). Those investigators arrived at a remarkable result. They had expected to regain estrogen responsive growth in the transfected hormone independent cells. This was definitely not the case. Instead, addition of E2 caused cell growth inhibition. Their results indicated that ERα was not a positive mediator, but instead a negative regulator. However, similarly transfected estrogen responsive cell lines such as MCF-7 and T47D were not E2 inhibited in those studies.
More recently, another estrogen receptor has been cloned and cDNA sequenced from rat prostate and ovary (Kuiper G G et al. (1996) Proc Natl Acad Sci USA 93, 5925-5930). It has now also been cloned from mouse (Tremblay G B et al. (1997) Mol Endocinol 11, 353-365) and human (Mosselman S et al. (1996) FEBS Lett 392, 49-53). This new receptor has been named estrogen receptor beta (ERβ). Evidence that ERβ is separate from ERα comes from the fact that the genes are located on different chromosomes (Enmark E et al. (1997) 82, 4258-4265). Therefore, ERβ is not simply an alternate splicing product of the ERα gene. Furthermore, ERβ is distinguishable from ERα based on critical differences in the amino acid sequences of functional domains (Kuiper G G et al. (1996) Proc Natl Acad Sci USA 93, 5925-5930; Enmark E et al. (1997) 82, 4258-4265; Dickson R B and Stancel G M (2000) J Natl Cancer Inst Monogr No. 27, 135-145). For example, the sequence homology between the two receptors is 97% in the DNA binding domain, but 59% in the C-terminal ligand-binding (i.e. steroid hormone-binding) domain, and only 17% in the N-terminal domain. The ERβ N-terminal domain is much abbreviated compared to the ERα (Enmark E et al. (1997) 82, 4258-4265). Rat ERβ contains an 18 amino acid insert in the domain binding the ligand. Despite the significant differences in structure, ERα and ERβ bind E2 with the same affinity (Kuiper G G et al. (1996) Proc Natl Acad Sci USA 93, 5925-5930; Dickson R B and Stancel G M (2000) J Natl Cancer Inst Monogr No. 27, 135-145). In fact, others (Tremblay G B et al. (1997) Mol Endocrinol 11, 353-365) have stated that ERβ has a slightly lower affinity for E2 than ERα (Tremblay G B et al. (1997) Mol Endocrinol 11, 353-365). Therefore, if either of these receptors mediates estrogen-induced growth, the steroid hormone concentrations required for one-half maximum growth (i.e. ED50), or for optimum growth (i.e. ED100), are expected to be about the same.
It is thought that ERα and ERβ are functionally interrelated (Kuiper G G et al. (1998) Endocrinology 139, 4252-4263) and that one role of ERβ is to modulate the transcriptional activity of ERα (Hall J M and McDonnell D P (1999) Endocrinology 140, 5566-5578). Clearly however, there are significant functional differences between ERα and ERβ, which have been discussed (Gustafsson J-Å (1999) J Endocrinol 163, 379-383). Also, there are functional differences expected because of the different pattern of steroid hormone binding shown by ERβ (Kuiper G G et al. (1996) Proc Natl Acad Sci USA 93, 5925-5930). For example, ERβ binds androgens whereas ERα does not. This fact, plus the location of ERβ in prostate indicates a new function that may be androgen related.
It should also be noted that there have been “estrogen related receptors” (ERR 1 and 2) or “orphan” receptors identified that share properties with ERα but do not have a known function and do not have a known ligand (Giguere V et al. (1988) Nature (Lond) 331, 91-94; Gustafsson J-Å (1999) J Endocrinol 163, 379-383). In fact, today, there are more than 70 “orphan” receptors seeking ligands and functions (Gustafsson J-Å (1999) Science (Wash D.C.) 284, 1285-1286).
The Secretory Immune System
Turning now to discussion of a separate body of work from that described above, as further background for understanding the present invention, it should be noted that the immunological function and physiological properties of the body's secretory immune system have been recognized for many years (Tomasi T B et al. (1965) J Exp Med 121, 101-124; Brandtzaeg P and Baklien K (1977) Ciba Foundation Symposium 46, 77-113; Tomasi T B (1970) Ann Rev Med 21, 281-298; Spiegelberg H L (1974) Adv Immunol 19, 259-294; Tomasi T B (1976) The Immune System of Secretions, Prentice-Hall, Englewood Clifts, N.J.; Mestecky J and McGhee J R (1987) Adv Immunol 40, 153-245). The major immunoglobulins secreted as mucosal immune protectors include IgA, IgM and IgG. In human serum, the percent content of IgG, IgA and IgM are 80, 6 and 13%, respectively. In humans, the major subclasses of IgG are IgG1, IgG2, IgG3 and IgG4. These are 66, 23, 7 and 4% of the total IgG, respectively. The relative content of human immunoglobulin classes/subclasses in adult serum follow the order IgG1>IgG2>IgA1>IgM>IgG3>IgA2>IgD>IgE (Spiegelberg H L (1974) Adv Immunol 19, 259-294). When the serum concentrations of immunoglobulins are compared to those in exocrine secretion fluids, the relative contents change dramatically (Brandtzaeg P (1983) Ann N.Y. Acad Sci 409, 353-382; Brandtzaeg P (1985) Scand J Immunol 22, 111-146). For example in colostrum (a breast fluid secretion), IgA is≧80% of the total immunoglobulins. IgM is≦10% of the total. IgG represents a few percent. In human colostrum and milk, IgG1 and IgG2 are the major subclasses of IgG (Kim K et al. (1992) Acta Paediatr 81, 113-118). Clearly, comparison of serum and mucosal fluid concentrations indicate selective immunoglobulin secretion.
Immunoglobulin Function. All human mucus membranes are protected by the secretory immune system (Hanson L Å and Brandtzaeg P (1989) In: Immunological Disorders in Infants and Children, 3rd edition, Stiehm E R, ed, Saunders, Philadelphia, pp 169-172). The primary protector is sIgA that is produced as dimers and larger polymers. A single joining “J” chain connects IgA monomers to form the dimers and polymers (Garcia-Pardo A et al. (1981) J Biol Chem 256, 11734-11738), and connects monomers of IgM to give pentamers (Niles M J et al. (1995) Proc Natl Acad Sci USA 92, 2884-2888). This critical joining endows these structures with a very important immunological property. IgA and IgM are known to bind to bacterial, parasite and viral surface antigens. These complexes bind to receptors on inflammatory cells leading to destruction of the pathogen by antibody-dependent cell-mediated cytotoxicity (Hamilton R G (1997) “Human Immunoglobulins” In: Handbook of Human Immunology, Leffell M S et al., eds, CRC Press, Boca Raton, Chapter 3). Dimeric and polymeric sIgA have a high antigen binding valence that effectively agglutinates/neutralizes bacteria and virus (Janeway C A Jr et al. (1999) Immunobiology, The Immune System in Health and Disease, 4th edition, Garland Publishing, New York, pp 326-327). Also, sIgA shows little or no complement activation. This means that it does not cause inflammatory responses (Johansen F E et al. (2000) Scand J Immunol 52, 240-248). In addition, the fact that IgA exists as two separate forms is significant (Loomes L M et al. (1991) J Immunol Methods 141, 209-218). The IgA1 predominates in the general circulation. In contrast, IgA2 is often higher in mucosal secretions such as those from breast, gut, and respiratory epithelium, salivary and tear glands, the male and female reproductive tracts, and the urinary tracts of both males and females. This difference in proportions is important to immune protection of mucosal surfaces. Although the secretory form of IgA1 is by and large resistant to proteolysis (Lindh E (1975) J Immunol 114, 284-286), a number of different bacteria secrete proteolytic enzymes that cleave it into Fab and Fc fragments (Wann J H et al. (1996) Infect Immun 64, 3967-3974; Poulsen K et al. (1989) Infect Immun 57, 3097-3105; Gilbert J V et al. (1988) Infect Immun 56, 1961-1966; Reinholdt J et al. (1993) Infect Immun 61, 3998-4000; Blake M S and Eastby C (1991) J Immunol Methods 144, 215-221; Burton J et al. (1988) J Med Chem 31, 1647-1651; Mortensen S B and Kilian M (1984) Infect Immun 45, 550-557; Simpson D A et al. (1988) J Bacteriol 170, 1866-1873; Blake M S and Swanson J et al. (1978) Infect Immun 22, 350-358; Labib R S et al. (1978) Biochim Biophys Acta 526, 547-559). In effect, the bacterial proteinases negate the neutralizing effects of multivalent sIgA1. In contrast, because of structural differences (Chintalacharuvu K R and Morrison S L (1996) J Immunol 157, 3443-3449), IgA2 lacks sites required for proteolysis. This makes IgA2 more resistant to bacterial digest than IgA1 (Hamilton R G (1997) “Human immunoglobulins” In: Handbook of Human Immunology, Leffell M S et al., eds, CRC Press, Boca Raton, Chapter 3).
With regard to IgM, its function is somewhat different. IgM antibodies serve primarily as efficient agglutinating and cytolytic agents. They appear early in the response to infection and are largely confined to the bloodstream. Whether secreted or plasma-borne, IgM is a highly effective activator of the classical complement cascade. It is less effective as a neutralizing agent or an effector of opsinization (i.e. facilitation of phagocytosis of microorganisms). Nonetheless, IgM complement activation causes lysis of some bacteria. The effects of the IgG class are more encompassing. All four subclasses cause neutralization, opsinization and complement activation to defend against mucosal microorganisms. IgG1 is an active subclass in this regard (Janeway C A Jr et al. (1999) Immunobiology, The Immune System in Health and Disease, 4th edition, Garland Publishing, New York, pp 326-327).
Immunoglobulin Structure. It was established that immunoglobulin A (IgA) represents 5 to 15% of the total plasma immunoglobulins in humans (Spiegelberg H L (1974) Adv Immunol 19, 259-294). IgA has a typical immunoglobulin four-chain structure (Mr 160,000) made up of two heavy chains (Mr 55,000) and two light chains (Mr 23,000) (Fallgreen-Gebauer E et al. (1993) Biol Chem Hoppe-Seyler 374, 1023-1028; Kratzin H et al. (1978) Hoppe-Seylers Z Physiol Chem 359, 1717-1745; Yang C et al. (1979) Hoppe-Seylers Z Physiol Chem 360, 1919-1940; Eiffert H et al. (1984) Hoppe-Seylers Z Physiol Chem 365, 1489-1495). In humans, there are two subclasses of IgA. These are IgA1 and IgA2 that have 1 and 2 heavy chains, respectively. The IgA2 subclass has been further subdivided into A2m(1) and A2m(2) allotypes (Mestecky J and Russell M W (1986) Monogr Allergy 19, 277-301; Morel A et al. (1973) Clin Exp Immunol 13, 521-528). IgA can occur as monomers, dimers, trimers or multimers (Lüllau E et al. (1996) J Biol Chem 271, 16300-16309). In plasma, 10% of the total IgA is polymeric while the remaining 90% is monomeric. Formation of dimeric or multimeric IgA requires the participation of an elongated glycoprotein of approximately Mr 15,000, designated the “J” chain (Mestecky J et al. (1990) Am J Med 88, 411-416; Mestecky J and McGhee J R (1987) Adv Immunol 40, 153-245; Cann G M et al. (1982) Proc Natl Acad Sci USA 79, 6656-6660). Structurally, the J chain is disulfide linked to the penultimate cysteine residue of heavy chains of two IgA monomers to form a dimeric complex of approximately Mr 420,000. The general structure of the dimer has been well described in the literature (Fallgreen-Gebauer E et al. (1993) Biol Chem Hoppe-Seyler 374, 1023-1028). Multimeric forms of IgA and IgM require only a single J chain to form (Mestecky J and McGhee J R (1987) Adv Immunol 40, 153-245; Chapus R M and Koshland M E (1974) Proc Natl Acad Sci USA 71, 657-661; Brewer J W et al. (1994) J Biol Chem 269, 17338-17348). The structures and chemical properties of IgA and IgM have been described in detail (Janeway C A Jr et al. (1996) Immunobiology, The Immune System in Health and Disease, Second edition, Garland Publishing, New York, pp 3-32 and pp 8-19).
Immunoglobulin Production. Dimeric and multimeric IgA and IgM are secreted by a number of exocrine tissues. IgA is the predominant secretory immunoglobulin present in colostrum, saliva, tears, bronchial secretions, nasal mucosa, prostatic fluid, vaginal secretions, and mucous secretions from the small intestine (Mestecky J et al. (1987) Adv Immunol 40, 153-245; Goldblum R M, et al. (1996) In: Stiehm E R, ed, Immunological Disorders in Infants and Children, 4th edition, Saunders, Philadelphia, pp 159-199; Heremans J F (1970) In: Immunoglobulins, Biological Aspects and Clinical Uses, Merler E, ed, National Academy of Sciences, Wash D.C. pp 52-73; Tomasi T B Jr (1971) In: Immunology, Current Knowledge of Basic Concepts in Immunology and their Clinical Applications, Good R A and Fisher D W, eds, Sinauer Associates, Stanford, Conn., p 76; Brandtzaeg P (1971) Acta Path Microbiol Scand 79, 189-203). IgA output exceeds that of all other immunoglobulins, making it the major antibody produced by the body daily (Heremans J F (1974) In: The Antigens, Vol 2, Sela M, ed, Academic Press, New York, pp 365-522; Conley ME et al. (1987) Ann Intern Med 106, 892-899. IgA is the major immunoglobulin found in human milk/whey/colostrum (Ammann A J et al. (1966) Soc Exp Biol Med 122, 1098-1113; Peitersen B et al. (1975) Acta Paediatr Scand 64, 709-717); Woodhouse L et al. (1988) Nutr Res 8, 853-864). IgM secretion is less abundant but can increase to compensate for deficiencies in IgA secretion.
During passage of IgA through the cell, its structure is modified. A Mr 80,000 fragment of the receptor containing all five of the extracellular domains becomes covalently attached to dimeric IgA to form secretory IgA (sIgA) (Fallgreen-Gebauer E et al. (1993) Biol Chem Hoppe-Seyler 374, 1023-1028). The receptor that mediates the translocation has been interchangeably called the “poly-Ig receptor” (poly-Ig receptor) or the “secretory component” (Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22, 2309-2315). For the purposes of the present disclosure, however, the term “poly-Ig receptor” refers to the full length Mr 100,000 transmembrane protein and the term “secretory component” denotes only the Mr 80,000 extracellular five domains of the receptor that become covalently attached to IgA in forming the sIgA structure (Fallgreen-Gebauer E et al. (1993) Biol Chem Hoppe-Seyler 374, 1023-1028; Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22, 2309-2315). Because of the unique structure of sIgA, it is highly resistant to acid and proteolysis (Lindh E (1975) J Immunol 114, 284-286) and therefore remains intact in secretions to perform extracellular immunological functions. IgM also binds secretory component, but not covalently (Lindh E and Bjork I (1976) Eur J Biochem 62, 271-278). However, IgM is less stabilized because of its different association with the secretory component, and therefore has a shorter functional survival time in acidic secretions (Haneberg B (1974) Scand J Immunol 3, 71-76; Haneberg B (1974) Scand J Immunol 3, 191-197).
The secretion mechanism for IgA and IgM are well described. Conversely, there is a fundamental question surrounding IgG secretion. There is no “J” chain present in IgG1 and IgG2. From the known facts of transcytosis/secretion of immunoglobulins (Johansen F E et al. (2000) Scand J Immunol 52, 240-248), it is unlikely that IgG secretion is mediated by the poly-Ig receptor. An epithelial receptor specific for IgG1 has been reported in bovine mammary gland (Kemler R et al. (1975) Eur J Immunol 5, 603-608). Apparently, it preferentially transports this class of immunoglobulins from serum into colostrum. Despite this 1975 report however, the receptor has not been chemically or structurally identified nor has the mechanism of transport of IgG monomers been satisfactorily defined. It is possible that this receptor is a member of a large group now designated as Fc receptors (Fridman W H (1991) FASEB J 5, 2684-2690), but there is one study with IgG showing that of 31 different long-term human carcinoma cell lines including breast “all lines were found to be consistently Fc receptor negative” (Kerbel R S et al. (1997) Int J Cancer 20, 673-679). One possible candidate for the epithelial transport of IgG1 is the neonatal Fc receptor (Raghavan M and Bjorkman P J (1996) Annu Rev Cell Dev Biol 12, 181-220). However, there is no indication yet of the presence of this receptor in adult mucosal tissues.
Transcytosis Mediating Receptors. J chain-containing IgA is produced and secreted by plasma B immunocytes located in the lamina propria just beneath the basement membrane of exocrine cells (Brandtzaeg P (1985) Scan J Immunol 22, 111-146). The secreted IgA binds to a Mr 100,000 poly-Ig receptor positioned in the basolateral surface of most mucosal cells (Heremans J F (1970) In: Immunoglobulins, Biological Aspects and Clinical Uses, Merler E, ed, National Academy of Sciences, Wash D.C., pp 52-73; Brandtzaeg P (1985) Clin Exp Immunol 44, 221-232; Goodman J W (1987) In: Basic and Clinical Immunology, Stites D P, Stobo J D and Wells J V, eds, Appleton and Lange, Norwalk, Conn., Chapter 4). The receptor-IgA complex is next translocated to the apical surface where IgA is secreted. The binding of dimeric IgA to the poly-Ig receptor is completely dependent upon the presence of a J chain (Brandtzaeg P (1985) Scan J Immunol 22, 111-146; Brandtzaeg P and Prydz H (1984) Nature 311:71-73; Vaerman J-P et al. (1998) Eur J Immunol 28, 171-182). Monomeric IgA will not bind to the receptor. The J chain requirement for IgM binding to the poly-Ig receptor is also true for this immunoglobulin (Brandtzaeg P (1985) Scan J Immunol 22, 111-146; Brandtzaeg P (1975) Immunology 29, 559-570; Norderhaug I N et al. (1999) Crit Rev Immunol 19, 481-508). Because IgA and IgM bind to the poly-Ig receptor via their Fc domains, and because of a repeating Ig-like structure in the extracellular domains, the poly-Ig receptor classifies as a member of the Fc superfamily of immunoglobulin receptors (Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22, 2309-2315; Daëron M (1997) Annu Rev Immunol 15, 203-234).
The poly-Ig receptor and the secretory component from human has been cDNA cloned and DNA sequenced (Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22, 2309-2315; Kraj{hacek over (c)}i P et al. (1995) Adv Exp Med Biol 371A, 617-623; Kraj{hacek over (c)}i P et al. (1991) Hum Genet 87, 642-648; Kraj{hacek over (c)}i P et al. (1989) Biochem Biophys Res Commun 237, 9-20) as has the poly-Ig receptor from mouse (Kushiro A and Sato T (1997) Gene 204, 277-282; Piskurich J F et al. (1995) and bovine tissue (Verbeet M P et al. (1995) Gene 164, 329-333). Altogether, the human poly-Ig receptor coding sequence encompassed 11 exons. The extracellular five domains originate from exons 3 (D1), exon 4 (D2) exon 5 (D3 and D4), exon 6 (D5), exon 7 (the conserved cleavage site to form the secretory component), exon 8 (the membrane spanning domain), exon 9 (a serine residue required for transcytosis), exon 9 (sequence to avoid degradation), exon 10, no known function) and exon 11 (sequence contains a threonine residue and the COOH terminus) (Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22, 2309-2315). With the exception of domains 3 and 4 (both from one exon), the receptor structure follows the rule of one domain/one exon. The poly-Ig receptor binds IgA and IgM via their Fc domains, and more particularly, via a specific amino acid sequence (15→37) of domain 1 (Bakos M-A et al. (1991) J Immunol 147, 3419-3426). Of the other extracellular domains, only D5 is known for a specific function. It contains the disulfide bonds that covalently attach to IgA to for sIgA during transcytosis. The role of this receptor in transcytosis of IgA/IgM has been well studied with mucosal tissues and epithelial cells in culture (Vaerman J P et al. (1998) Eur J Immunol 28, 171-182; Fahey J V et al. (1998) Immunol Invest 27, 167-180; Brandtzaeg P (1997) J Reprod Immunol 36, 23-50; Loman S et al. (1997) Am J Physiol 272, L951-L958; Mostov K E et al. (1995) Cold Spring Harbor Symp Quant Biol 60, 775-781; Schaerer E et al. (1990) J Cell Biol 110, 987-998).
During passage of IgA through the cell, its structure is modified. A Mr 80,000 fragment of the receptor containing all five of the extracellular domains becomes covalently attached to dimeric IgA to form secretory IgA (sIgA) (Fallgreen-Gebauer E et al. (1993) Biol Chem Hoppe-Seyler 374, 1023-1028). The receptor that mediates the translocation has been interchangeably called the “poly-Ig receptor” (poly-Ig receptor) or the “secretory component” (Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22, 2309-2315). For the purposes of the present disclosure, however, the term “poly-Ig receptor” refers to the full length Mr 100,000 transmembrane protein and the term “secretory component” denotes only the Mr 80,000 extracellular five domains of the receptor that become covalently attached to IgA in forming the sIgA structure (Fallgreen-Gebauer E et al. (1993) Biol Chem Hoppe-Seyler 374, 1023-1028; Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22, 2309-2315). Because of the unique structure of sIgA, it is highly resistant to acid and proteolysis (Lindh E (1975) J Immunol 114, 284-286) and therefore remains intact in secretions to perform extracellular immunological functions. IgM also binds secretory component, but not covalently (Lindh E and Bjork I (1976) Eur J Biochem 62, 271-278). However, IgM is less stabilized because of its different association with the secretory component, and therefore has a shorter functional survival time in acidic secretions (Haneberg B (1974) Scand J Immunol 3, 71-76; Haneberg B (1974) Scand J Immunol 3, 191-197).
While the secretion mechanism for IgA and IgM are well described, conversely, a fundamental question surrounds IgG secretion. There is no “J” chain present in IgG1 and IgG2. From the known facts of transcytosis/secretion of immunoglobulins (Johansen F E et al. (2000) Scand J Immunol 52, 240-248), it is unlikely that IgG secretion is mediated by the poly-Ig receptor. An epithelial receptor specific for IgG1 has been reported in bovine mammary gland (Kemler R et al. (1975) Eur J Immunol 5, 603-608). Apparently, it preferentially transports this class of immunoglobulins from serum into colostrum. Despite this 1975 report however, the receptor has not been chemically or structurally identified nor has the mechanism of transport of IgG monomers been satisfactorily defined. It is possible that this receptor is a member of a large group now designated as Fc receptors (Fridman W H (1991) FASEB J 5, 2684-2690), but there is one study with IgG showing that of 31 different long-term human carcinoma cell lines including breast “all lines were found to be consistently Fc receptor negative” (Kerbel R S et al. (1997) Int J Cancer 20, 673-679). One possible candidate for the epithelial transport of IgG1 is the neonatal Fc receptor (Raghavan M and Bjorkman P J (1996) Annu Rev Cell Dev Biol 12, 181-220). However, there is no indication yet of the presence of this receptor in adult mucosal tissues.
Fc receptors are so named because they bind specific heavy chains (Fc domains). However, before coming to this conclusion, it should be emphasized strongly that the Fc family represented by Fcγ (IgG), Fcα (IgA), and Fcμ (IgM) have traditionally been considered to be located on lymphoid series cells (Fridman WH (1991) FASEB J 5, 2684-2690; Raghavan M and Bjorkman PJ (1996) Annu Rev Cell Dev Biol 12, 181-220). There is only limited experimental support for the concept that these receptors are located on epithelial cells (Tonder O et al. (1976) Acta Pathol Microbiol Scand 84, 105-111). For the family of leukocyte IgG receptors, 12 transmembrane or soluble receptor isoforms are known. These are grouped into three classes FcγR1(CD64), Fcγ RII (CD32) and Fcγ RIII (CD16) (Valerius T et al. (1997) Blood 90, 4485-4492). For IgA, there is one gene that encodes several receptors) (i.e. Fcα) by alternate splicing to yield forms from Mr 55,000 to 110,000 (Pleass RJ et al. (1996) Biochem J 318, 771-777; van Dijk TB et al. (1996) Blood 88, 4229-4238; Morton HC et al. (1996) Immunogenetics 43, 246-247). One of these, FcαR1 is constitutively expressed on monocytes and macrophages and other leukocytes. It binds IgA1 and IgA2 with about the same affinity. The receptor for IgM (i.e.Fcμ) is less well defined, but still has been partially characterized as a Mr 60,000 protein present on activated B cells and other B series cells (Ohno T et al. (1990) J Exp Med 172, 1165-1175). The Fc superfamily has another very important aspect pertinent to this disclosure. Receptors of this family mediate negative effects on cells (Cambier JC (1997) Proc Natl Acad Sci USA 94, 5993-5995). These receptors have an intracellular amino acid sequence motif I/VxYxxL (SEQ ID NO:1 and SEQ ID NO:2) described as an immunoreceptor tyrosine-based inhibitory motif (ITIM) that signals cell growth shutdown after ligand binding. These signals have been characterized in the FCγRIIB1 receptors of human and mouse (Olcese L et al. (1996) J Immuno 156, 4531-4534). The hallmark of these ITIM receptors is that they shut off growth factor dependent growth.
Although the advances and teachings in the prior art have indicated that a serum-borne inhibitor of steroid hormone responsive tumor cell growth exists, until now there has been no adequate isolation or identification of such an inhibitor, and very little understanding of its mode of action has been gained. There is no satisfactory in vitro testing model presently available for demonstrating steroid hormone responsive cell growth that can be correlated to the in vivo situation, or for testing drugs, or other natural or synthetic substances for possible hormone-mimicking or anti-hormone effects.